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K. Berger, J. Kramme, R. W. Beuerman, C. J. R. Sheppard, J. F. Bille; Depth Related Decay and Imaging Depth of the Second Harmonic Signal in the Rabbit Eye. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5672.
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© ARVO (1962-2015); The Authors (2016-present)
Second Harmonic Generation (SHG) is well established for imaging collagen tissues in the human body with the potential of developing into an in-vivo method. Considering the applicability of SHG as a clinical examination modality, one major question is the penetration depth of the SHG signal and therefore the effective imaging depth, especially in the backward direction. The present work has investigated this issue using the anterior segment of the rabbit eye.
Prior dissected limbus, sclera and cornea samples were shock frozen, mounted in OCT and subsequently sectioned into various thicknesses (100 to 300 µm), parallel to the surface of the eye using a cryostat. Tissue slices along with the intact unprocessed tissue pieces were imaged using an Olympus IX71, FV300 and a Ti:Sapphire (Coherent Mira) operating at 830 nm and constant input power. Five frames were averaged with a total acquisition time of 5 seconds. One image covered an area of 700x700 µm. SHG was detected in both forward and backward (F-B) directions. Image data was further processed in Matlab.
The depth related decrease of SHG in limbus, sclera and cornea followed an exponential decay. The imaging depths into each of the tissues agreed in the forward and backward directions. Comparing the different tissue types, the imaging depth was greatest in the cornea, even though the signal was weak compared with that from limbus and sclera. The effective attenuation depth as well as the observed increase in the F/B ratio showed that the backward directed SHG signal decays considerably faster than in the forward direction.
For in vivo imaging, a major limiting factor concerning the effective imaging depth with SHG is the generation of the signal. We find that other factors such as absorption of the second harmonic signal seem to play no major role. As only the backward directed signal is potentially useful for in vivo diagnostic procedures, the different appearance of the collagen bundles in the backward direction and the strong decay of the signal along with the high F/B ratios of the regarded ocular tissues might limit the general applicability of SHG in this context.
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